Production of chemicals from microorganisms has been an important application of biotechnology. Typically, the step in developing such a bio-production method may include 1) selection of a proper microorganism host, 2) elimination of metabolic pathways leading to by-products, 3) deregulation of such pathways at both enzyme activity level and the transcriptional level, and 4) overexpression of appropriate enzymes in the desired pathways. The present invention has employed combination of the steps above to redirect carbon flow from phenylalanine to tyrosine through phenylalanine hydroxylase which supplies the necessary precursor and energy for the desired biosynthesis of Para-hydroxycinnamic.
Para-hydroxycinnamic (PHCA) is a useful monomer for production of Liquid Crystal Polymers (LCP). LCP's may be used in electronic connectors and telecommunication and aerospace applications. LCP resistance to sterilizing radiation has also enabled these materials to be used in medical devices as well as chemical, and food packaging applications.
Para-hydroxycinnamic (PHCA) or p-coumarate is a known intermediate in the lignin biosynthetic pathway in plants (Plant Biochemistry, Ed. P. M. Dey, Academic Press, 1997). Methods of isolation and purification of PHCA are known (R. Benrief, et al., Phytochemistry, 1998, 47, 825-832; WO 972134). These methods are time consuming and cumbersome and a more facile method of production is needed for the large scale synthesis of this monomer. A fermentation route offers one possible solution.
A fermentation route to PHCA will require the engineering of several of the key enzymes involved in PHCA synthesis into an appropriate host. PHCA is a natural intermediate in the lignin biosynthetic pathway and key enzymes for synthesis may be obtained from a variety of plants. Lignin biosynthesis is initiated by the conversion of phenylalanine into cinnamate through the action of phenylalanine ammonia lyase (PAL). The second enzyme of the pathway is cinnamate-4-hydroxylase (C4H), a cytochrome P450-dependent monooxygenase (P450) which is responsible for the conversion of cinnamate to PHCA also called p-coumarate.
Thus, it is evident that one possible route to PHCA is via phenylalanine ammonia lyase (PAL) from phenylalanine. However this route also requires the presence of the second enzyme, cinnamate-4-hydroxylase, an enzyme which is rare in most microbes.
Information available indicates that PAL from some plants and micro-organisms can accept tyrosine as substrate in addition to its ability to convert phenylalanine to cinnamate. In such reactions the enzyme activity is designated tyrosine ammonia lyase (TAL). Conversion of tyrosine by TAL results in the direct formation of PHCA from tyrosine without the intermediacy of cinnamate. However, all natural PAL/TAL enzymes prefer to use phenylalanine rather than tyrosine as their substrate. The level of TAL activity is always lower than PAL activity, but the magnitude of this difference varies over a wide range. For example, the parsley enzyme has a KM for phenylalanine of 15-25 μM and for tyrosine 2.0-8.0 mM with turnover numbers 22/sec and 0.3/sec respectively (Appert et al., Eur. J. Biochem. 225:491 (1994)). In contrast, the maize enzyme has a KM for phenylalanine only fifteen times higher than for tyrosine, and turnover numbers about ten-fold higher (Havir et al., Plant Physiol. 48:130 (1971)). The exception to this rule, is the yeast, Rhodosporidium, in which a ratio of TAL catalytic activity to PAL catalytic activity is approximately 0.58 (Hanson and Havir in The Biochemistry of Plants; Academic: New York, 1981; Vol. 7, pp 577-625). Thus an alternate pathway to PHCA, might involve the direct conversion of tyrosine to PHCA via TAL, assuming an abundant source of tyrosine. Tyrosine is however, generally in low supply in most microorganisims, whereas phenylalanine is abundant. A method to convert phenylalanine to tyrosine would facilitate the pathway to PHCA through TAL.
Phenylalanine hydroxylase (PAH) systems appear to be infrequent in prokaryotes. Phenylalanine hydroxylase has been reported in a few species belonging to the α division of the class Proteobacteria and in Pseudomonas aeruginosa in, the γ division (Zhao et al., Proc. Natl. Acad. Sci. USA. 91: 1366 (1994)). Of these, Pseudomonas aeruginosa is the best characterized at the molecular-genetic level (Song et al. Mol. Microbiol. 22:497-507 (1996) and Zhao et al., Proc. Natl. Acad. Sci. USA. 91: 1366 (1994)). Pseudomonas aeruginosa possesses a multi-gene operon that includes phenylalanine hydroxylase which is homologous with mammalian phenylalanine hydroxylase, tryptophan hydroxylase, and tyrosine hydroxylase (Zhao et al., Proc. Natl. Acad. Sci. USA. 91: 1366 (1994)). The bacterial Phenylalanine hydroxylase from Pseudomonas aeruginosa and Chromobacterium violaceum has been cloned, expressed, purified, and fully characterized (Abu-Omar et al. Book of Abstracts, 219 ACS National Meeting, San Francisco, Calif., Mar. 26-30, (2000) INOR-068 Publisher: American Chemical Society, Washington, D.C.)). Moreover, the presence of PAH in Streptomyces aureofaciens has been demonstrated (Maladkar, Hind. Antibiot. Bull., 28: 1-4, 30-6 (1986)).
The enzymatic conversion of phenylalanine to tyrosine is known in eukaryotes. Human phenylalanine hydroxylase is specifically expressed in the liver to convert L-phenylalanine to L-tyrosine (Wang et al. J. Biol. Chem. 269 (12): 9137-46 (1994)). Deficiency of the PAH enzyme causes classic phenylketonurea, a common genetic disorder.
The literature is silent as to the conversion of glucose to para-hydroxycinnamic acid via re-directing the carbon flow from phenylalanine to tyrosine through phenylalanine hydroxylase maximizing the concentration of tyrosine in the cells to allow for the use of the TAL pathway for conversion of glucose to PHCA.
The problem to be solved here is to develop an industrially suitable method for production of tyrosine and para-hydroxycinnamic acid using genetically engineered microorganisms.
Applicants have solved the stated problem by engineering several recombinant microorganisms comprising at least one gene encoding a phenylalanine hydroxylase activity and at least one gene encoding a phenylalanine and tyrosine ammonium lyase activity for the production of tyrosine and para-hydroxycinnamic acid from fermentable carbon substrates.